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Introduction:

DNA, Deoxyribonucleic acid, is a double stranded, helical nucleic

acid molecule which determines inherited structure of a protein. The

?steps? are made of bases: adenine, guanine, cytosine, and thymine. The

sides are sugar and phosphate molecules. Restriction enzymes are

enzymes that cut DNA at restriction sites, leaving fragments blunt or

sticky. The restriction fragments are separated using a technique called

gel electrophoresis.

DNA has a negative charge so when an electrical charge is

applied it makes DNA move to the positive side. DNA is placed in

agarose gel. Smaller fragments move faster. The purpose of this lab is to

separate DNA fragments using gel electrophoresis. Hind III cuts AAGCTT

between the two irst A?s. EcoRI cuts at GAATTC between the G and the

A. Hind III and EcoRI both make sticky ends.

Results:

Our results for this lab were EcoRI separated into five fragments.

Hind III separated into four fragments. The control only had one fragment.

(See chart A and figure 1-1 for distances)

Discussion:

The purpose of this lab was to see how gel electrophoresis

separates DNA fragments. We used Hind III, EcoRI, and a controlled

enzyme. Some fragments were hard to see because of smearing. These

were the bigger fragments. Loading the DNA was difficult and if you

weren?t careful you could rupture the wells which ruined the lab. We,

fortunately, did not run into this problem.

Abstract:

The purpose of this lab is to separate DNA fragments with gel

electrophoresis using EcoRI and Hind III. Restriction enzymes are used to

break up the DNA, then negatively charged DNA is placed in a gel

casting tray. Then it is placed into an electrophoresis chamber. An

electrical field is placed across the agarose gel which forces the

fragments to move down the gel. The amount of lines show how many

fragments there is in the DNA. We had five fragments for EcoRI and six

for Hind III. The no enzyme had only one fragment.

Procedures:

We sealed the ends of a gel casting tray with masking tape and

inserted a comb into the slots. The tray was filled about 6mm high with

agarose gel. It covered half the height of the comb. We waited ten

minutes for the gel to solidify. Then we placed the tray in a gel box and

made sure that the comb was at a negative (black) end. The box was

filled with tris-borate-EDTA buffer so it covered the entire surface of the

gel. The combs were removed without ripping the wells. The micro pipet

was used to load the lambda EcoRI, lambda Hind III, and lambda only

into the wells. We dipped the pipet trough the surface of the buffer over

the wells and expelled the contents. The top of the electrophoresis

chamber was closed and electrical leads were connected. The dye was

observed as it moved shortly after the power supply was turned on. The

power supply was turned off after the bands migrated near the end of

the gel and the top of the electrophoresis chamber was removed. We

removed the gel from the gel casting tray and examined it under a light

box and compared it to the ideal gel (figure1-2).

:

Restriction Enzymes: Cleavage of DNA lab

University of Illinois. (1999). Experiment 2

Gel Electrophoresis of DNA. In

Molecular Biology Cyberlab, online:

Http://www.life.uluc.edu/molbio/geldigest/electro.html


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